Our overall goals are to understand how eukaryotic cells, mammalian tissues and animals (including people) protect against the cytotoxic, mutagenic and carcinogenic effects of alkylating agents present in our endogenous and exogenous environment. Alkylating agents are present as both naturally occurring and synthetic compounds. In addition they are commonly used in the cancer clinic for chemotherapy. Given that one in three people will be diagnosed with cancer during their lifetime (at least in the developed world) large numbers of people will be deliberately exposed to these toxic agents during chemotherapy. To gain an understanding of how eukaryotic cells respond to alkylating agents (and other agents) we have employed S. cerevisiae, cultured mammalian cells, and mouse model systems. We will continue to use these organisms in the next funding period. Three Specific Aims are proposed, all of which represent a natural progression from a subset of our achievements in the previous funding period. Part of the first Specific Aim represents a continuation of the Genomic Phenotyping approach to understanding responses of eukaryotic cells to damaging agents;this was a new approach that we developed for screening a collection of ~ 4,800 S. cerevisiae strains, each deleted for a different non-essential gene. We now propose to extend this study to include the remaining 1,200 essential genes. Moreover we propose to extend this genomic scale analysis of yeast by systematically interrogating the subcellular localization of ~ 4,000 yeast proteins upon exposure to damaging agents. The second Specific Aim is designed to take the insights gained from our genomic studies of how S. cerevisae cells respond to damaging agents, and determine whether these insights are relevant in human cells. Specifically, we have found that in addition to DMArepair and cell cycle checkpoints, there are a number of hitherto unexpected cellular pathways that play equally important roles in helping cells recover from exposure to carcinogenic DMAdamaging agents. We aim to establish whether these pathways are similarly important in human cells. Finally, in the third Specific Aim we will explore the notion that more DNA repair is not necessarily beneficial. In previous funding cycles we have found that imbalances within and between DNA repair pathways can cause increases in spontaneous mutation, and can sometimes lead to cells being sensitive to DNA damaging agents. More recently we found that expression of the human AAG 3-methyadenine DNA glycosylase in yeast and human cells creates a genomic instability phenotype. We will further explore the mechanism by which this instability is induced, and we will generate transgenic mice expressing AAG (and the mouse counterpart, Aag) to determine whether such an imbalance in DNA repair creates a cancer prone phenotype. The health relatedness of this project lies in the fact that it will contribute to our understanding of Cancer Etiology.